U.S. patent number 5,403,714 [Application Number 08/005,086] was granted by the patent office on 1995-04-04 for method for in vitro detection of formed elements in biological samples.
Invention is credited to Paul N. Fiedler, Robert A. Levine, Stephen C. Wardlaw.
United States Patent |
5,403,714 |
Levine , et al. |
April 4, 1995 |
Method for in vitro detection of formed elements in biological
samples
Abstract
A biologic sample such as feces, sputum, cervical tissue,
pleural fluids, exudates, cytologic specimens, or the like, is
tested for the presence or absence of: ova; parasites;
microorganisms; inflammatory, neoplastic tissue cells; or other
target materials which are indicative of infestation, disease or
infection. The sample is mixed with a buffer fluid and placed in a
transparent tube which contains a volume-constricting cylindrical
insert for gravimetric separation of components of the sample. The
mixture is centrifuged, and the annular space between the insert
and tube bore is examined under magnification for the presence of
the target materials.
Inventors: |
Levine; Robert A. (Guilford,
CT), Wardlaw; Stephen C. (Old Saybrook, CT), Fiedler;
Paul N. (New Haven, CT) |
Family
ID: |
25129129 |
Appl.
No.: |
08/005,086 |
Filed: |
January 15, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
783397 |
Oct 28, 1991 |
5252460 |
Oct 12, 1993 |
|
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Current U.S.
Class: |
435/7.2;
435/287.2; 435/288.1; 435/7.21; 435/7.22; 435/7.23; 436/165;
436/172; 436/536; 436/541; 436/63; 436/64; 436/70; 436/805;
436/810; 436/813; 436/824 |
Current CPC
Class: |
G01N
33/569 (20130101); G01N 33/56966 (20130101); G01N
33/574 (20130101); Y10S 436/824 (20130101); Y10S
436/813 (20130101); Y10S 436/80 (20130101); Y10S
436/81 (20130101); Y10S 436/805 (20130101) |
Current International
Class: |
G01N
33/569 (20060101); G01N 33/574 (20060101); G01N
033/536 (); G01N 033/558 () |
Field of
Search: |
;435/7.2,7.21,7.22,7.23,296,973 ;422/57,58
;436/63,64,66,70,165,172,514,536,541,800,805,810,813,824
;73/61.43,61.51,61.72,149,444 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Scheiner; Toni R.
Assistant Examiner: Chin; Christopher L.
Attorney, Agent or Firm: Jones; William W.
Parent Case Text
This is a continuation of copending patent application Ser. No.
07/783,397, filed Oct. 28, 1991, now U.S. Pat. No. 5,352,460,
granted Oct. 12, 1993.
Claims
What is claimed is:
1. A method for testing a tissue sample for the presence or absence
of abnormal cell morphology, said method comprising the steps
of:
a) providing a transparent sample-receiving tube containing a
volume-restricting generally cylindrical insert positioned
substantially coaxially with a tube axis, said insert being
operable to produce a restricted volume annulus in the tube
adjacent a tube side wall;
b) providing an abnormal cell-highlighting reagent in the tube for
visually differentiating the abnormal cells from remaining
constituents of the sample;
c) forming a mixture of the tissue sample and a non-biological
liquid buffer, which buffer will allow gravimetric separation of
abnormal cell structures from other formed components in the
mixture;
d) substantially filling said tube with said mixture;
e) centrifuging said filled tube to gravimetrically separate any
abnormal cells from other formed components in the mixture; and
f) examining said annulus under magnification to determine the
presence or absence of abnormal cell morphology in said
annulus.
2. The method of claim 1 wherein said reagent is acridine
orange.
3. The method of claim 1 wherein said reagent comprises abnormal
cell-specific antibodies coupled with fluorophore.
4. The method of claim 1 wherein said reagent comprises abnormal
cell agglutinating antibodies.
5. A method for performing a cytological examination of a
biological sample to detect the presence or absence of cancer
cells, said method comprising the steps of:
a) providing a transparent sample-receiving tube containing a
volume-restricting generally cylindrical insert positioned
substantially coaxially with a tube axis, said insert being
operable to produce a restricted volume annulus in the tube
adjacent a tube side wall;
b) providing a cancer cell-highlighting reagent in the tube for
visually differentiating the cancer cells from remaining
constituents of the sample;
c) forming a mixture of the biological sample and a non-biological
liquid buffer, which buffer will allow gravimetric separation of
cancer cells from other formed components in the mixture;
d) substantially filling said tube with said mixture;
e) centrifuging said filled tube to gravimetrically separate any
cancer cells from other formed components in the mixture; and
f) examining said annulus under magnification to determine the
presence or absence of cancer cells in said annulus.
6. The method of claim 5 wherein said reagent is acridine
orange.
7. The method of claim 5 wherein said reagent comprises cancer
cell-specific antibodies coupled with fluorophore.
8. The method of claim 5 wherein said reagent comprises cancer cell
agglutinating antibodies.
9. The method of claim 5 wherein said biological sample comprises
cervical cells.
Description
TECHNICAL FIELD
This invention relates to the identification of target materials in
biological samples, which materials are indicative of disease,
parasite infestation, or the like. More particularly, this
invention relates to the detection of ova; parasites, either free
living or in cysts; macroorganisms; neoplastic or inflammatory
tissue cells, all in biological samples, such as feces, pleural
fluids, exudates, cytologic specimens, or the like.
BACKGROUND ART
Biological samples are routinely examined for the presence of
abnormal organisms or cells, such as ova, parasites,
microorganisms, and inflammatory cells by conventional light
microscopy of smear preparations. Visual detection of such
materials in smears is hindered by the presence of masking
particulate or other matter interspersed between cells.
Additionally, standard smear preparations utilize only a minute
fraction of the sample since the smears must be thin enough to
allow the passage of light. Examination of multiple smears to
compensate for these drawbacks is impractical in the busy hospital
laboratory. Consequently, the sensitivity of disease detection is
suboptimal using the smear methodology.
Ova and parasites present in stool may be concentrated by employing
filtration techniques whereby the sample specimen is made
relatively dense so that the ova, and/or parasites, and/or cysts
will float on the surface of the sample. This technique however
does not permit the visualization of the target organisms or ova in
the concentrated specimen. The target layer must be aspirated or
transferred to a slide for examination, whereupon a large number of
the target organisms or cysts are lost.
Cytologic evaluation of tissue cells in smears derived from
biological fluids and tissues for the purpose of diagnosing cancer
is an integral part of the practice of medicine. These examinations
are; however, typically performed on less than 5% of the cells
sampled due to the aforesaid requirement that smears be thin.
Furthermore, as noted, tissue cells in these preparations are often
obscured from view by contaminating blood cells and debris.
Cytologic evaluation of tissue cells in feces for the diagnosis of
colon cancer has not been used to any significant extent. In fact,
the index of a recently published authoritative cytology textbook
(Comprehensive Cytopathology, Marluce Bibbo; ed. W. B. Saunders
Company, 1991) contains no references to either stool or feces
evaluation. This deficit may be explained by the paucity of tissue
cells in the standard stool smear, and the abundance of obscuring
debris. Consequently, cytologic examination of stool by the smear
protocol is virtually useless as part of a cancer screening.
Medical diagnostics of stool and other biological samples would be
greatly improved by a technique which does not depend on the use of
a very limited amount of sample material, such as is inherent in
the examination of specimen smears. The reason for this is that
when one is able to use a larger specimen sample in the screening
or examination, the odds are increased that target materials will
be included in the sample and identified.
U.S. Pat. No. 4,190,328 granted Feb. 26, 1980 to R. A. Levine et al
describes a process for the detection of blood-borne parasites in a
sample of centrifuged anticoagulated whole blood. The blood sample
is mixed with a stain and then centrifuged in a tube containing a
volume-restricting float. Target parasites which may be in the
blood sample are highlighted by the stain and trapped between the
float and the bore of the tube where they can be visually observed
under appropriate magnification.
U.S. Pat. No. 4,717,660 granted Jan. 5, 1988 to T. H. Schulte
discloses a procedure for detecting bacteria in an anticoagulated
blood sample, and for assessing phagocytic activity in the blood
sample. The blood sample is mixed with a stain that will highlight
the bacteria, and the mixture is centrifuged in a tube containing a
volume-restricting float. The buffy coat in the centrifuged blood
sample settles into the restricted space between the tube bore and
float, and the cell bands in the buffy coat are thereby physically
elongated. Any bacteria in the blood will be differentially stained
and will be located in one of the buffy coat cell bands, where such
bacteria can be observed under appropriate magnification.
The aforesaid patented procedures are restricted to diagnosing
blood samples, and do not suggest any diagnostic procedures wherein
non-fluid biological samples, or fluid biological samples, other
than blood, can be analysed quickly and dependably for target
malady-specific materials.
Disclosure of the Invention
This invention relates to the detection of target malady-specific
materials in fluid and non-fluid biological specimen samples.
Target materials detectable by this invention include: parasites;
parasite ova; parasite cysts; microorganisms; neoplastic or
inflammatory tissue cells; and the like. Specimen samples which can
be examined using this invention include: feces; cytological
specimens; pleural fluids; exudates; and the like.
The examination of the specimen is made in a transparent tube which
contains an insert operable to limit the available volume in the
tube into which the target materials will settle upon
centrifugation of the sample in the tube. The tube can be a
capillary tube, or, if necessary, it can be larger in volume for
examining certain biological specimens, such as feces. The insert
in the tube is generally cylindrical and is made from a plastic
material having a specific gravity which ensures that the target
materials, if present, will settle into the annular free space
between the insert and the tube bore wall. In most cases, a
specific gravity which ensures that the insert will sink during
centrifugation to the bottom of the specimen sample tube will
suffice for proper performance of the invention; however, in some
cases it may be desirable to have the insert "float on" or "settle
into" one of the centrifuged component layers. Stains or other
colorants may be incorporated into the tube in a dry coating on the
tube bore, to be released when the specimen is added to the tube.
When non-liquid or non-fluid specimens are being examined, or when
minute amounts of a fluid specimen are being examined, a liquid
medium will be added to the specimen tube in order to provide an
environment in the tube in which the target materials, if present,
will be separated and isolated from the rest of the specimen. When
larger preevacuated centrifuge tubes and inserts are used, as
described in copending U.S. patent application No. 579,274, filed
Sep. 5, 1990 by Levine and Wardlaw, all necessary liquid reagents
or buffers may be preloaded in the tube.
This method for the detection of ova, cysts, parasites, or other
microorganisms, inflammatory and tissue cells in stool and other
biological samples is far more sensitive than conventional smears.
The increased sensitivity derives from the ability to analyze a
large sample volume (1 ml) of a sample suspension rapidly and
accurately. More than one thousand conventional smears
corresponding to approximately six hundred man-hours would be
required for light microscopy examination of a 1 ml sample
volume.
This invention utilizes centrifugation to concentrate the
particulate matter in feces and other biologic samples at the
bottom or the top of a glass tube. The target ova, parasites, other
microorganisms, tissue cells, or the like in the sample form a
separate discrete layer. Targets in the layer are pressed against
the side of the tube by the plastic insert which has the same
specific gravity as the targets. The insert is of appropriate
dimensions to spread these target elements in a thin film along the
sides of the tube thereby bringing the targets into the focal plane
of a fluorescent microscope. Acridine orange present in the tube
stains the DNA of cells green and the RNA red. The metachromatic
fluorescent staining allows the identification of the targets, and,
in the case of assay for cancer cells, enables the classification
of epithelial and other tissue cells as benign or malignant. The
juxtaposition of cells of the same class facilitates the comparison
of nuclear and cytoplasmic features between cells, a process which
is essential to the diagnosis of malignancy.
It is therefore an object of the invention to provide an improved
technique for assaying a biological specimen sample for target
malady-specific materials.
It is a further object of the invention to provide a technique of
the character described wherein a relatively large volume of the
biological sample can be assayed quickly and easily, while
providing accurate data.
It is yet another object of the invention to provide a technique of
the character described wherein the biological sample being assayed
can be a non-fluid material, such as feces.
It is an additional object to provide a technique of the character
described wherein the presence of cancer cells in the biological
sample can be detected.
These and other objects and advantages of the invention will become
more readily apparent from the following detailed description of
several preferred embodiments of this invention when taken in
conjunction with the accompanying drawings in which:
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is side elevational view of a centrifuge tube assembly for
use in performing the procedure of this invention; and
FIG. 2 is a view of the tube assembly of FIG. 1 showing a
centrifuged specimen sample therein, and with the target-containing
area in the tube assembly being blown up in size to point out the
nature of the invention.
DETAILED DESCRIPTION OF THE BEST MODE
Referring now to FIGS. 1 and 2, there is shown in FIG. 1, a tube 2,
which may be a glass capillary tube, or other transparent tube, and
which contains an insert 4 made of a plastic, which has a specific
gravity that causes the insert 4 to settle through the biological
sample mixture 5 to the bottom 6 of the tube 2, when the latter is
centrifuged with the biological sample therein. Any fibrous or
other solid components in the sample may settle into a clump 7 in
the bottom of the tube 2 and the targets form a band T in the
annulus between the insert 4 and tube 2. A plastic cap 10 closes
the bottom 6 of the tube 2 prior to centrifugation. In certain
cases, as when feces is being examined, the insert 4 may imbed
itself in the clump 7.
The following describes a general procedure for preparing
biological samples for analysis in accordance with this invention,
and a number of specific examples of sample analyses are also set
forth.
In each of the examples described hereinafter, the biological
sample was mixed with a buffer solution consisting of Dulbecco's
phosphate buffered saline with 0.15% EDTA. The bottom of the tube
was plugged with 4% agarose (1 cm) and a plastic cap. Samples were
spun in the tube, along with an acridine orange stain and the
plastic insert, for five minutes at 12,000 rpm prior to visual
inspection by fluorescent microscopy at 500x power. The tubes used
in the following examples were microhematocrit or capillary tubes,
however, larger tubes and inserts can be used when larger samples
need to be inspected. Other buffer solutions and dyes or stains can
be used depending on the nature of the samples being assayed.
EXAMPLE 1
One gm of fresh solid stool, which was intentionally altered by the
addition thereto of parasites to simulate infestation, was placed
in 3 ml of buffer. The mixture was vortexed until homogeneous, and
the centrifuge tube was filled with an aliquot of the homogeneous
mixture. After centrifugation, particulate material was seen to
layer out at the bottom of the tube and the plastic insert was
embedded in the particulate matter layer. Lighter constituents were
trapped between the insert and tube bore. These lighter trapped
constituents were examined under a fluorescent microscope, and were
found to include ova, parasites, granulocytes, epithelial cells,
bacterial and yeast. The aforesaid examination revealed that the
procedure can detect in stool both parasite infestation and other
cell types that may be indicative of disease or infection.
EXAMPLE 2
One ml of ascites fluid obtained by paracentesis was centrifuged to
form a loose pellet. Ten ul of the formed pellet was placed in 1 ml
of the buffer to which mixture 20 ul of whole blood (EDTA), to
duplicate a naturally bloody sample, and to which buccal epithelial
scrapings were added in order to increase potential target cells,
whereby the applicability of the invention to performing cervical
PAP tests could be confirmed. The resultant mixture was vortexed
briefly to blend the constituents homogeneously, and the centrifuge
tube with insert was then filled with a portion of the resultant
mixture. After centrifugation, the plastic insert extended from the
bottom of the tube through all cell layers. Upon microscopic
examination, it was determined that the nontarget formed debris
consisting of erythrocytes, followed by granulocytes, monocytes,
and lymphocytes were layered out at the bottom. The top layer was a
mixture of target epithelial cells, mesothelial cells and
platelets. Excellent separation of blood and non-blood cells was
achieved, as is necessary in cytological analysis. The
concentration of cells between the insert and tube wall enabled
easy differentiation of normal and abnormal cells.
EXAMPLE 3
Cells obtained by swabbing the endo and ectocervical mucosae of a
fresh cone biopsy specimen were placed in 1 ml of the buffer, to
which 1 ml of a mucolytic agent (0.1 gm N-acetyl cysteine in 10 ml
sodium citrate diluted 3:4 with water to 300 m OSM) was added. The
mixture was vortexed briefly to form a homogeneous sample, and a
capillary tube was filled with a portion of the mixed sample. The
filled tube was then centrifuged and examined. The insert extended
through all cell layers in the sample. Fluorescent microscopy
revealed highly dysplastic squamous cells and atypical endocervical
cells in the area between the insert and tube. Cancer cells can
thus be readily observed in the sample using the procedure of this
invention. Additionally, more subtle cell changes can be identified
using the invention. For example, atypical stage cells, ie, cells
which are in the process of transforming from a normal to a
cancerous morphology, are readily identified using the procedure of
this invention.
Various target-highlighting reagents such as acridine orange stain;
antibodies coupled with fluorophore; or polyvalent
target-agglutinating antibodies can be included in the tube in
order to render the targets more readily observable. The
agglutinating antibodies can be used to bind targets together
whereby they will be observed in a better defined band in the
annulus.
Since many changes and variations of the disclosed embodiments of
the invention may be made without departing from the inventive
concept, it is not intended to limit the invention otherwise than
as required by the appended claims.
* * * * *